LAUSR

Transport of membrane and cytosolic proteins into the primary cilium is essential for its role in cellular signaling. Using a virtual 3D super-resolution light microscopy, the movements of membrane and soluble proteins from the cytoplasm to the primary cilium was mapped. In addition to the well-characterized intraflagellar transport (IFT) route, we found two new pathways within the lumen of the primary cilium were identified. Specifically, passive diffusion and vesicle-assisted transport routes that, are adopted by proteins for cytoplasm-cilium transport in live cells. Through these pathways, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route, and more than half of membrane-embedded G protein coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside primary cilia. Furthermore, ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signal-dependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins. [Media: see text] [Media: see text] [Media: see text] [Media: see text].